Kit for detecting the ApoE4 allele, and for diagnosing the existence or risk of developing Alzheimer&#39;s disease

ABSTRACT

Methods of diagnosing or prognosing Alzheimer&#39;s disease in a subject are disclosed. The methods involve directly or indirectly detecting the presence or absence of an apolipoprotein E type 4 (ApoE4) isoform or DNA encoding ApoE4 in the subject. The presence of ApoE4 indicates the subject is at risk of developing Alzheimer&#39;s disease. A novel immunochemical assay for detecting the presence or absence of the Apoliprotein E (ApoE) E4 allele in a subject is also disclosed.

This invention was made with Government support under NIH LEAD Award5R35 AGO 7922 and NIH Alzheimer's Disease Research Center 5P50 AGO 5128.The Government has certain rights to this invention.

RELATED APPLICATIONS

This application is a divisional of prior continuation-in-partapplication Ser. No. 08/227,044, filed on Apr. 13, 1994, now U.S. Pat.No. 5,508,167, which is a continuation-in-part of prior application Ser.No. 08/114,448; filed Aug. 31, 1993, now abandoned; which is acontinuation-in-part application of Ser. No. 07/959,992, filed Oct. 13,1992, now abandoned entitled METHODS OF DETECTING ALZHEIMER'S DISEASE,the disclosure of which is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to methods of diagnosing and prognosingAlzheimer's disease. The invention further includes immunologicalmethods of distinguishing various isoforms of apolipoprotein E (ApoE),and particularly for distinguishing ApoE4 from ApoE2 and ApoE3.

BACKGROUND OF THE INVENTION

The senile plaque and congophilic angiopathy are abnormal extracellularstructures found in abundance in brain of patients with Alzheimer'sdisease. The biochemical composition of these structures has beenextensively studied to better understand their possible role in thepathogenesis of this dementing disease. The mature senile plaque is acomplex structure, consisting of a central core of amyloid fibrilssurrounded by dystrophic neurites, axonal terminals and dendrites,microglia and fibrous astrocytes. See D. Selkoe Neuron 6,487-498 (1991).The amyloid core of the senile plaque surrounding blood vessels,producing the congophilic angiopathy, is a peptide of 39 to 43 aminoacids termed the β-Amyloid (Aβ) peptide. G. Glenner and C. Wong,Biochem. Bioshys. Res. Comm. 120, 885-890 (1984). Aβ peptide is found inbrain in Alzheimer's disease, Down's syndrome, hereditary cerebralhemorrhage of the Dutch type, and in old age. K. Kosik, Science 256,780-783 (1992). Aβ is produced by abnormal proteolytic processing of alarger protein, the amyloid precursor protein (APP). See K. Bayreutherand C. Masters, Brain Path. 1, 241-251 (1991).

The senile plaque and congophilic angiopathy contain proteins inaddition to βA peptide. APP itself, among others, has been identified inthe senile plaque by histochemical studies employing antibodiesrecognizing either the amino- and carboxy-termini of the precursorprotein. See, e.g., F. Tagliavine et al., Neurosci. Lett. 128, 117-120(1991); C. Joachim et al., Amer. Jour. Path. 138, 373-384 (1991); Themechanisms by which these proteins aggregate in the extracellular spaceto associate with the senile plaque and congophilic angiopathy are notknown.

Apolipoprotein E (ApoE) performs various functions as a proteinconstituent of plasma lipoproteins, including its role in cholesterolmetabolism. It was first identified as a constituent ofliver-synthesized very low density lipoproteins which function in thetransport of triglycerides from the liver to peripheral tissues. Thereare three major isoforms of ApoE, referred to as ApoE2, ApoE3 and ApoE4which are products of three alleles at a single gene locus. Threehomozygous phenotypes (Apo-E2/2, E3/3, and E4/4) and three heterozygousphenotypes (ApoE3/2, E4/3 and E4/2) arise from the expression of any twoof the three alleles. The most common phenotype is ApoE3/3 and the mostcommon allele is E3. See Mahley, R. W., Science 240:622-630 (1988).

The amino acid sequences of the three types differ only slightly. ApoE4differs from ApoE3 in that in ApoE4 arginine is substituted for thenormally occurring cysteine at amino acid residue 112. The most commonform of ApoE2 differs from ApoE3 at residue 158, where cysteine issubstituted for the normally occurring arginine. See Mahley, Science,supra.

While there has been considerable research into the mechanismsunderlying Alzheimer's disease, there continues to be an ongoing needfor new ways to investigate and combat this disorder.

SUMMARY OF THE INVENTION

Methods of diagnosing or prognosing Alzheimer's disease in a subject aredisclosed. One embodiment comprises detecting the presence or absence ofDNA encoding an apolipoprotein E type 4 (ApoE4) isoform in the subject.Another embodiment comprises detecting the presence or absence of anApoE4 isoform. The presence of an ApoE4 isoform or of DNA encoding anApoE4 isoform indicates that the subject is afflicted with Alzheimer'sdisease or at risk of developing Alzheimer's disease. Kits fordiagnosing or prognosing Alzheimer's disease are also described.

The immunochemical assay comprises collecting a sample such as a bloodsample from the subject, optionally combining the sample with a reducingagent, contacting the sample to a solid support which specifically bindsreactive sulfhydryl groups, then separating the sample from the solidsupport, and then detecting by immunoassay either (i) the presence orabsence of ApoE in said sample, the presence of ApoE in said sampleindicating the subject has at least one allele for ApoE4, the absence ofApoE in said sample indicating said subject has no alleles for ApoE4, or(ii) the presence or absence of ApoE immobilized on the solid support,the presence of ApoE immobilized on said solid support indicating saidsubject has one or no alleles for ApoE4, the absence of ApoE immobilizedon the solid support indicating said subject has two alleles for ApoE4.

A further aspect of the present invention is a kit useful for detectingthe presence of the ApoE4 allele in a subject by immunochemical assay.The kit comprises a solid support which specifically binds reactivesulfhydryl groups, and antibodies which specifically bind ApoE.

A simple test is provided to determine whether an individual has theApoE4 isoform, and whether the individual is homozygous for ApoE4 (i.e.produces only ApoE4) or is heterozygous for ApoE4 (i.e. produces ApoE4and an additional isoform of ApoE). The test is based on the differencesin the amino acid sequences of the three major ApoE isoforms, ApoE2,ApoE3, and ApoE4. ApoE4 contains no cysteines, ApoE3 contains onecysteine, and ApoE2 contains two cysteines. By relying on chemical meansfor distinguishing among the different ApoE isoforms, the problem ofpotential cross-reactivity of antibodies used to carry out theimmunoassay with multiple ones of the isoforms is obviated.

The foregoing and other objects and aspects of the present invention areexplained in detail in the specification set forth hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, top panel, shows proteins in cerebrospinal fluid which bind toimmobilized βA peptide, and to a control peptide (the "even-hydropeptide", or "E.H.") after incubation and: elution with phosphatebuffered saline, column 1; elution with 5% sodium dodecyl sulfate,column 2; elution with 4 molar urea, column 3; or elution with 6 molarguanidine hydrochloride, column 4. Binding of Apolipoprotein E is shownby immunohistochemical staining in the bottom panel.

FIG. 2, top panel, shows the binding of proteins in cerebrospinal fluidto various immobilized peptides. Binding of Apolipoprotein E is againshown by immunohistochemical staining in the bottom panel.

FIG. 3 shows the age at onset for subjects with 0, 1, and 2 ApoE4alleles. Each curve is labeled by the number of ApoE4 alleles. Thesymbol `*` indicates multiple diagnoses within a short interval. Onsetcurves were estimated by Kaplan-Meier product limit distributions andwere clearly distinct (logrank chi--square=53.8, 2 degrees of freedom,p<0.0001).

FIG. 4 shows the results of an assay of the present invention, where"load" refers to coupling buffer plus ApoE sample prior to combiningwith resin, and "elute" refers to the supernatant recovered from theassay after centrifugation.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides a method of screening(e.g., diagnosing or prognosing) for Alzheimer's disease in a subject.The method comprises detecting the presence or absence of ApoE4 isoformor of DNA encoding an ApoE4 isoform in the subject. The presence of suchisoform or DNA indicates that the subject is afflicted with Alzheimer'sdisease or at risk of developing Alzheimer's disease. Suitable subjectsinclude those which have not previously been diagnosed as afflicted withAlzheimer's disease, those which have previously been determined to beat risk of developing Alzheimer's disease, and those who have beeninitially diagnosed as being afflicted with Alzheimer's disease whereconfirming information is desired. For example, patients diagnosed ordetermined to be afflicted with dementia, particularly patients who hadpreviously been clinically normal who are determined to be afflictedwith a progressive dementia, are suitable subjects. Thus, the presentinvention may be employed in detecting both familial Alzheimer's disease(late onset and early onset) as well as sporadic Alzheimer's disease.Many Alzheimer's disease patients encountered in practice have noobvious family history and have been classified as sporadic. However,genetic factors in early--and late--onset of familial Alzheimer'sdisease (FAD) are well documented. Late-onset Alzheimer's disease is theclassification usually used if the disease is diagnosed to occur afterthe age of 65 in humans.

Observing whether or not the ApoE4 isoform of ApoE is present or absentin a subject enables one to observe or determine whether or not asubject is afflicted with or at increased risk of developing Alzheimer'sdisease. Affliction with the disease is more likely if ApoE4 is present.A subject with ApoE4 is at increased risk of developing Alzheimer'sdisease over subjects in which ApoE4 is absent. A subject who is "atincreased risk of developing Alzheimer's disease" is one who ispredisposed to the disease, has genetic susceptibility for the diseaseor is more likely to develop the disease than subjects in which ApoE4 isabsent.

Further, the methods of the present invention can be used to aid indetermining the prognosis of a subject afflicted with or at risk forAlzheimer's disease based on the observation of how many alleles forApoE4 are detected in the subject. The subject's prognosis is morenegative if the presence of ApoE4 is detected than if it is absent; thesubject's prognosis is most negative if the presence of more than oneallele for ApoE4 is detected.

For example, subjects with the ApoE4/4 genotype are as much as eighttimes as likely to be affected by Alzheimer's disease as subjects withthe ApoE2/3 or ApoE3/3 genotypes. Further, the average age of onset ofAlzheimer's disease and the average age of survival is younger for thosehaving one ApoE4 allele, and youngest for those having two ApoE4alleles. Thus, a subject's prognosis for Alzheimer's disease is morelikely to be negative if the subject has an ApoE4 allele and mostnegative if the subject has more than one ApoE4 allele. The negativeprognosis can be viewed in terms of increased likelihood of developingthe disease, or of increased likelihood of developing the disease ordying at an earlier age.

It is preferred and contemplated that the methods described herein beused in conjunction with other clinical diagnostic information known ordescribed in the art which are used in evaluation of subjects withAlzheimer's disease or suspected to be at risk for developing suchdisease.

ApoE phenotypes and genotypes are well described and known in the art asdescribed above. The established nomenclature system as well as thephenotypes and genotypes for ApoE, are described in, for example, Zanniset al., J. Lipid. Res. 23:911 et seq. (1982), which is incorporated byreference herein.

The step of detecting the presence or absence of ApoE4 or of DNAencoding such isoform (including the number of alleles for ApoE4) may becarried out either directly or indirectly by any suitable means. Avariety of techniques are known to those skilled in the art. Allgenerally involve the step of collecting a sample of biological materialcontaining either DNA or ApoE from the subject, and then detectingwhether or not the subject possesses ApoE4 or DNA encoding such isoformfrom that sample. For example, the detecting step may be carried out bycollecting an ApoE sample from the subject (for example, fromcerebrospinal fluid, or any other fluid or tissue containing ApoE), andthen determining the presence or absence of an ApoE4 isoform in the ApoEsample (e.g., by isoelectric focusing or immunoassay).

The isolation and characterization of ApoE is described, for example, inRall et al., Methods in Enzymology 128:273-287 (1986), Davignon et al.,Arteriosclerosis 8:1-21 (1988), and in Warnick et al., Clin. Chem.25:279-284 (1979), all of which are incorporated by reference herein.Isoelectric focusing is an electrophoretic technique by which themolecules are separated based on their isoelectric points (pI) along acontinuous pH gradient. Reference proteins, commercially available(e.g., Sigma Chemical Company, St. Louis, Mo.), are used to indicate agradient along which the sample proteins match up according to wheretheir pH matches their pI. In Warnick, et al. very-low-densityapolipoproteins are isolated from plasma samples and applied toisoelectric focusing gels and the isoelectric focusing patterns of theApoE isoforms are obtained. According to the Warnick et al. procedure,pI values of the ApoE isoforms, E2, E3 and E4, were about 5.9, 6.0 and6.1 respectively in 8 M/L urea at 4° C. See also, Pagnan et al., J.Lipid. Res. 18:613-622 (1977) and Utermann et al., FEBS Lett. 56:352-355(1975), both of which are incorporated by reference herein. Variousisoeletric focusing-type techniques are also provided in Rall et al.supra, including analytical isoelectric focusing, cysteamine treatment,neuraminidase treatment, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, as well as amino acid analysis and nucleic acidsequence analysis, and capillary isoelectric focusing is described in H.Swartz, Bio/Technology 12, 408-09 (April 1994).

In the alternative, the detecting step may be carried out by collectinga biological sample containing DNA from the subject, and thendetermining the presence or absence of DNA encoding an ApoE4 isoform inthe biological sample. Any biological sample which contains the DNA ofthat subject may be employed, including tissue samples and bloodsamples, with blood cells being a particularly convenient source. Theamino acid sequences and nucleic acid sequences for ApoE2, ApoE3 andApoE4 are known and described. See, for example, Paik et al., Proc.Nat'l Acad. Sci. USA 82:3445-3449 (1985), incorporated by referenceherein, for the nucleic acid sequence of ApoE3 and ApoE4; and Mahley,Science, supra for the amino acid sequence information.

Determining the presence or absence of DNA encoding an ApoE4 isoform maybe carried out with an oligonucleotide probe labelled with a suitabledetectable group, or by means of an amplification reaction such as apolymerase chain reaction or ligase chain reaction (the product of whichamplification reaction may then be detected with a labelledoligonucleotide probe or a number of other techniques). Further, thedetecting step may include the step of detecting whether the subject isheterozygous or homozygous for the gene encoding an ApoE4 isoform.Numerous different oligonucleotide probe assay formats are known whichmay be employed to carry out the present invention. See, e.g., U.S. Pat.No. 4,302,204 to Wahl et al.; U.S. Pat. No. 4,358,535 to Falkow et al.;U.S. Pat. No. 4,563,419 to Ranki et al.; and U.S. Pat. No. 4,994,373 toStavrianopoulos et al. (applicants specifically intend that thedisclosures of all U.S. Patent references cited herein be incorporatedherein by reference).

Amplification of a selected, or target, nucleic acid sequence may becarried out by any suitable means. See generally D. Kwoh and T. Kwoh,Am. Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplificationtechniques include, but are not limited to, polymerase chain reaction,ligase chain reaction, strand displacement amplification (see generallyG. Walker et al., Proc. Natl. Acad. Sci. USA 89, 392-396 (1992); G.Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992)),transcription-based amplification (see D. Kwoh et al., Proc. Natl. AcadSci. USA 86, 1173-1177 (1989)), self-sustained sequence replication (or"3SR") (see J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874-1878(1990)), the Qβ replicase system (see P. Lizardi et al., BioTechnology6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or"NASBA") (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), therepair chain reaction (or "RCR") (see R. Lewis, supra), and boomerangDNA amplification (or "BDA") (see R. Lewis, supra). Polymerase chainreaction is currently preferred.

DNA amplification techniques such as the foregoing can involve the useof a probe, a pair of probes, or two pairs of probes which specificallybind to DNA encoding ApoE4, but do not bind to DNA encoding ApoE2 orApoE3 under the same hybridization conditions, and which serve as theprimer or primers for the amplification of the ApoE4 DNA or a portionthereof in the amplification reaction (likewise, one may use a probe, apair of probes, or two pairs of probes which specifically bind to DNAencoding ApoE2, but do not bind to DNA encoding ApoE3 or ApoE4 under thesame hybridization conditions, and which serve as the primer or primersfor the amplification of the ApoE2 DNA or a portion thereof in theamplification reaction; and one may use a probe, a pair of probes, ortwo pairs of probes which specifically bind to DNA encoding ApoE3, butdo not bind to DNA encoding ApoE2 or ApoE4 under the same hybridizationconditions, and which serve as the primer or primers for theamplification of the ApoE3 DNA or a portion thereof in the amplificationreaction).

In general, an oligonucleotide probe which is used to detect DNAencoding ApoE4 is an oligonucleotide probe which binds to DNA encodingApoE4, but does not bind to DNA encoding ApoE2 or ApoE3 under the samehybridization conditions. The oligonucleotide probe is labelled with asuitable detectable group, such as those set forth below in connectionwith antibodies. Likewise, an oligonucleotide probe which is used todetect DNA encoding ApoE2 is an oligonucleotide probe which binds to DNAencoding ApoE2 but does not bind to DNA encoding ApoE3 or ApoE4 underthe same hybridization conditions, and an oligonucleotide probe which isused to detect DNA encoding ApoE3 is an oligonucleotide probe whichbinds to DNA encoding ApoE3 but does not bind to DNA encoding ApoE2 orApoE4 under the same hybridization conditions.

Polymerase chain reaction (PCR) may be carried out in accordance withknown techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188. In general, PCR involves, first, treating anucleic acid sample (e.g., in the presence of a heat stable DNApolymerase) with one oligonucleotide primer for each strand of thespecific sequence to be detected under hybridizing conditions so that anextension product of each primer is synthesized which is complementaryto each nucleic acid strand, with the primers sufficiently complementaryto each strand of the specific sequence to hybridize therewith so thatthe extension product synthesized from each primer, when it is separatedfrom its complement, can serve as a template for synthesis of theextension product of the other primer, and then treating the sampleunder denaturing conditions to separate the primer extension productsfrom their templates if the sequence or sequences to be detected arepresent. These steps are cyclically repeated until the desired degree ofamplification is obtained. Detection of the amplified sequence may becarried out by adding to the reaction product an oligonucleotide probecapable of hybridizing to the reaction product (e.g., an oligonucleotideprobe of the present invention), the probe carrying a detectable label,and then detecting the label in accordance with known techniques, or bydirect visualization on a gel. When PCR conditions allow foramplification of all ApoE allelic types, the types can be distinguishedby hybridization with allelic specific probe, by restrictionendonuclease digestion, by electrophoresis on denaturing gradient gels,or other techniques. A PCR protocol for determining the ApoE genotype isdescribed in Wenham et al., The Lancet 337:1158-1159 (1991),incorporated by reference herein. Examples of primers effective foramplification and identification of the ApoE isoforms are describedtherein. Primers specific for the ApoE polymorphic region (whetherApoE4, E3 or E2) can be employed. In Wenham, for example, PCR primersare employed which amplify a 227 bp region of DNA that spans the ApoEpolymorphic sites (codons 112 and 158, which contain nucleotides 3745and 3883). The amplified fragments are then subjected to restrictionendonuclease CfoI which provides different restriction fragments fromthe six possible ApoE genotypes which may be recognizable on anelectrophoresis gel. See also, Hixon et al., J. Lipid Res. 31:545-48(1990); Houlston et al., Hum. Genet. 83:364-365 (1989) Wenham et al.,Clin. Chem. 37:241-244 (1991); and Konrula et al., 36:2087-92 (1990) foradditional methods, all of which are incorporated by reference herein.

Ligase chain reaction (LCR) is also carried out in accordance with knowntechniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general,the reaction is carried out with two pairs of oligonucleotide probes:one pair binds to one strand of the sequence to be detected; the otherpair binds to the other strand of the sequence to be detected. Each pairtogether completely overlaps the strand to which it corresponds. Thereaction is carried out by, first, denaturing (e.g., separating) thestrands of the sequence to be detected, then reacting the strands withthe two pairs of oligonucleotide probes in the presence of a heat stableligase so that each pair of oligonucleotide probes is ligated together,then separating the reaction product, and then cyclically repeating theprocess until the sequence has been amplified to the desired degree.Detection may then be carried out in like manner as described above withrespect to PCR.

It will be readily appreciated that the detecting steps described hereinmay be carried out directly or indirectly. Thus, for example, if eitherApoE2 or ApoE3 is also detected in the subject, then it is determinedthat the subject is not homozygous for ApoE4; and if both ApoE2 andApoE3 are detected in the subject, then it is determined that thesubject is neither homozygous nor heterozygous for ApoE4. Other means ofindirectly determining allelic type could be by measuring polymorphicmarkers that are linked to ApoE allele, as has been demonstrated for theVNTR (variable number tandem repeats) and the ApoB alleles (Decorter etal., DNA & Cell Biology 9(6), 461-69 (1990).

As an alternative to isoeiectric focusing and techniques for alleledetection, the step of determining the presence or absence of the ApoE4isoform in a sample may be carried out by an antibody assay with anantibody which selectively binds to ApoE4 (i.e., an antibody which bindsto ApoE4 but exhibits essentially no binding to ApoE2 or ApoE3 in thesame binding conditions). When one wishes to determine the precise ApoEcomplement of a patient and whether or not that patient is homozygous orheterozygous for ApoE4, then antibodies which selectively bind to ApoE2and ApoE3 may also be employed (i.e., an antibody which binds to ApoE2but exhibits essentially no binding to ApoE3 or ApoE4 in the samebinding conditions; an antibody which binds to ApoE3 but exhibitsessentially no binding to ApoE2 or ApoE4 in the same bindingconditions).

Antibodies used to selectively or specifically bind ApoE2, ApoE3, andApoE4 can be produced by any suitable technique. For example, monoclonalantibodies may be produced in a hybridoma cell line according to thetechniques of Kohler and Milstein, Nature 265, 495-97 (1975). ApoE2,ApoE3, or ApoE4 may be obtained from a human patient determined to behomozygous therefore, then purified by the technique described in S.Rall et al., Methods in Enzymol. 128, 273 (1986), and used as theimmunogen for the production of monoclonal or polyclonal antibodies.Purified ApoE isoforms may be produced by recombinant means to express abiologically active isoform, or even an immunogenic fragment thereof maybe used as an immunogen. Monoclonal Fab fragments may be produced inEscherichia coli from the known sequences by recombinant techniquesknown to those skilled in the art. See, e.g., W. Huse, Science 246,1275-81 (1989) (recombinant Fab techniques); P. Wenham et al., Lancet337, 1158 (1991) (ApoE PCR primers). The DNA encoding one subtype ofApoE can be obtained and converted to the other by site-directedmutagenesis. See, e.g., T. Kunkel et al., Methods in Enzymol. 154,367-382 (1987); T. Kunkel, U.S. Pat. No. 4,873,192.

The term "antibodies" as used herein refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodiesmay be monoclonal or polyclonal and may be of any species of origin,including (for example) mouse, rat, rabbit, horse, or human, or may bechimeric antibodies, and include antibody fragments such as, forexample, Fab, F(ab')₂, and Fv fragments, and the corresponding fragmentsobtained from antibodies other than IgG.

For this invention, an antibody selectively or specifically binding ApoEor a particular ApoE isoform (ligand) generally refers to a moleculecapable of reacting with or otherwise recognizing or binding such aligand. An antibody has binding affinity for a ligand or is specific fora ligand if the antibody binds or is capable of binding the ligand asmeasured or determined by standard antibody-antigen or ligand-receptorassays, for example, competitive assays, saturation assays, or standardimmunoassays such as ELISA or RIA. This definition of specificityapplies to single heavy and/or light chains, CDRs, fusion proteins orfragments of heavy and/or light chains, that are specific for the ligandif they bind the ligand alone or in combination.

Antibody assays (immunoassays) may, in general, be homogeneous assays orheterogeneous assays. In a homogeneous assay the immunological reactionusually involves the specific antibody, a labeled analyte, and thesample of interest. The signal arising from the label is modified,directly or indirectly, upon the binding of the antibody to the labeledanalyte. Both the immunological reaction and detection of the extentthereof are carried out in a homogeneous solution. Immunochemical labelswhich may be employed include free radicals, radioisotopes, fluorescentdyes, enzymes, bacteriophages, coenzymes, and so forth.

In a heterogeneous assay approach, the reagents are usually thespecimen, the antibody of the invention and a system or means forproducing a detectable signal. Similar specimens as described above maybe used. The antibody is generally immobilized on a support, such as abead, plate or slide, and contacted with the specimen suspected ofcontaining the antigen in a liquid phase. The support is then separatedfrom the liquid phase and either the support phase or the liquid phaseis examined for a detectable signal employing means for producing suchsignal. The signal is related to the presence of the analyte in thespecimen. Means for producing a detectable signal include the use ofradioactive labels, fluorescent labels, enzyme labels, and so forth. Forexample, if the antigen to be detected contains a second binding site,an antibody which binds to that site can be conjugated to a detectablegroup and added to the liquid phase reaction solution before theseparation step. The presence of the detectable group on the solidsupport indicates the presence of the antigen in the test sample.Examples of suitable immunoassays are the radioimmunoassay,immunofluorescence methods, enzyme-linked immunoassays, and the like.

Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022 to Skold et al. titled "Methods for ModulatingLigand-Receptor Interactions and their Application," U.S. Pat. No.4,659,678 to Forrest et al., U.S. Pat. No. 4,376,110 to David et al.,U.S. Pat. No. 4,275,149 to Litman et al., U.S. Pat. No. 4,233,402 toMaggio et al., and U.S. Pat. No. 4,230,767 to Boguslaski et al.

Antibodies which selectively bind an ApoE isoform (i.e., bind to one ofApoE2, ApoE3 or ApoE4 while showing essentially no binding to the otherunder the same binding conditions) may be conjugated to a solid supportsuitable for a diagnostic assay (e.g., beads, plates, slides or wellsformed from materials such as latex or polystyrene) in accordance withknown techniques, such as precipitation. Antibodies which bind an ApoEisoform may likewise be conjugated to detectable groups such asradiolabels (e.g., ³⁵ S, ¹²⁵ I, ¹³¹ I), enzyme labels (e.g., horseradishperoxidase, alkaline phosphatase), and fluorescent labels (e.g.,fluorescein) in accordance with known techniques.

Kits for determining if a subject is or was afflicted with or is or wasat increased risk of developing Alzheimer's disease will include atleast one reagent specific for detecting for the presence or absence ofApoE4 and instructions for observing that the subject is or wasafflicted with or is or was at increased risk of developing Alzheimer'sdisease if the presence of ApoE4 is detected. The kit may optionallyinclude a nucleic acid for detection of the ApoE4 gene or instructionsfor isoelectric focusing methods for detecting ApoE4.

Diagnostic kits for carrying out antibody assays may be produced in anumber of ways. In one embodiment, the diagnostic kit comprises (a) anantibody which binds ApoE2, ApoE3, or ApoE4 conjugated to a solidsupport and (b) a second antibody which binds ApoE2, ApoE3, or ApoE4conjugated to a detectable group. The reagents may also includeancillary agents such as buffering agents and protein stabilizingagents, e.g., polysaccharides and the like. The diagnostic kit mayfurther include, where necessary, other members of the signal-producingsystem of which system the detectable group is a member (e.g., enzymesubstrates), agents for reducing background interference in a test,control reagents, apparatus for conducting a test, and the like. Asecond embodiment of a test kit comprises (a) an antibody as above, and(b) a specific binding partner for the antibody conjugated to adetectable group. Ancillary agents as described above may likewise beincluded. The test kit may be packaged in any suitable manner, typicallywith all elements in a single container along with a sheet of printedinstructions for carrying out the test.

Immunochemical Methods for Detecting ApoE4

The novel immunochemical methods disclosed here can be easily carriedout to detect and distinguish ApoE alleles in a subject for any purposedescribed herein or otherwise, whether screening subjects (e.g.,diagnosing or prognosing) for Alzheimer's disease or screening subjectsfor cardiovascular disease.

The immunochemical assay comprises optionally (but preferably) combininga sample such as a blood sample collected from the subject with areducing agent, then contacting the sample to a solid support whichspecifically binds reactive sulfhydryl groups, then separating thesample from the solid support; and then detecting by immunoassay either(i) the presence or absence of ApoE in said sample, the presence of ApoEin said sample indicating the subject has at least one allele for ApoE4,the absence of ApoE in said sample indicating said subject has noalleles for ApoE4, or (ii) the presence or absence of ApoE immobilizedon the solid support, the presence of ApoE immobilized on said solidsupport indicating said subject has one or no alleles for ApoE4, theabsence of ApoE immobilized on the solid support indicating said subjecthas two alleles for ApoE4.

Any sample of biological material containing ApoE may be used. Forexample, the sample may be collected from blood, blood serum, bloodplasma, cerebrospinal fluid, or any other fluid or tissue containingApoE from the subject.

It will be readily appreciated that the detecting steps described hereinmay be carried out directly or indirectly. Thus, for example, if eitherApoE2 or ApoE3 is also detected in the subject, then it is determinedthat the subject is not homozygous for ApoE4; and if both ApoE2 andApoE3 are detected in the subject, then it is determined that thesubject is neither homozygous nor heterozygous for ApoE4. If eitherApoE2 or ApoE3 is present the possibility exists that the individual isheterozygous for ApoE4. Heterozygosity for ApoE4 is also indicated ifthe amount of ApoE4 bound to the column is determined to be about twicethe amount eluted from the column. Alternatively, to make furtherdistinctions, the assay of the present invention can be used as aninitial screen and combined with another assay capable of distinguishingbetween ApoE2, ApoE3, and ApoE4, such as an immunoassay, isoelectricfocusing, or PCR analysis of DNA encoding ApoE2, ApoE3, and ApoE4.

Any solid support which specifically binds reactive sulfhydryl groupsmay be employed in carrying out the present invention. By specific, itis meant that the solid support will covalently bind sulfhydryl groupsin the presence of competing groups such as amino, hydroxyl, andcarboxylate. These solid supports include, but are not limited to, solidsupports employing tresyl chemistry (K. Nilsson, Methods. Enzymol. 63,56 (1984)), activated gels having pyridyl disulfide (T. Egoroy et al.,Proc. Natl. Acad. Sci. USA 72, 171 (1975)) or dithio-5-nitrobenzoic acidmoieties that produce disulfide linkages, and TNB-thiol(5-thio-2-nitrobenzoic acid) agarose gels (e.g., Pierce product number20409G, available from Pierce Chemical Co., Post Office Box 117,Rockford, Ill. 61105 USA). Preferred solid supports are thoserepresented by the formula R-COCH₂ X, wherein R is a solid substratesuch as a gel and X is halogen (e.g., Cl, Br, I, preferably I).Particularly preferred is the SULFOLINK® coupling gel available asproduct numbers 44895G, 20405G, 20401G, 20402G, and 20403G from Pierce,Post Office Box 117, Rockford, Ill. 61105 USA.

Any agent capable of reducing the disulfide bond in cysteine residues tothe corresponding reactive sulfhydryl groups may be used as the reducingagent to carry out the present invention. Preferably the reducing agentis a lower molecular weight thiol, more preferably mercaptoethanol,dithiothreitol, and mercaptoethylamine. Reduction of the disulfide bondsin cysteines will typically be carried out at slightly alkaline anddenaturing conditions (for example, 8M urea or 6M guanidine HCl).

Once the sample has been contacted to the solid support, it is separatedfrom the solid support by any known means and, if desired, collected forfurther testing.

The immunoassay step is carried out by specifically binding ApoE with anantibody which specifically binds to ApoE. The antibodies include alltypes of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE producedby any known suitable method as described above.

An advantage of the foregoing technique is that it is not necessary toemploy an antibody which binds specifically to one ApoE isoform withoutbinding to one or more of the other isoforms. However, suchisoform-specific antibodies may be employed if desired. Antibodiesspecific for ApoE are readily made and are known.

Immunoassays may, in general, be homogeneous assays or heterogeneousassays, as described above. As also described above, antibodies whichspecifically bind ApoE may be conjugated to a solid support suitable fora diagnostic assay (e.g., beads, plates, slides or wells formed frommaterials such as latex or polystyrene) in accordance with knowntechniques, and they may likewise be conjugated to detectable groups.

Diagnostic kits for carrying out the immunochemical assay may beproduced in a number of ways. In one embodiment, the kit comprises (a) asolid support which specifically binds reactive sulfhydryl groups, (b)an antibody which binds to ApoE2, ApoE3, and/or ApoE4, and (c),optionally, a reducing agent. In another embodiment, the diagnostic kitcomprises (a) an antibody which binds ApoE2, ApoE3, and/or ApoE4conjugated to a solid support and (b) a second antibody which bindsApoE2, ApoE3, and/or ApoE4 conjugated to a detectable group. Thereagents may also include ancillary agents such as buffering agents andprotein stabilizing agents, e.g., polysaccharides and the like. Thediagnostic kit may further include, where necessary, other members ofthe signal-producing system of which system the detectable group is amember (e.g., enzyme substrates), agents for reducing backgroundinterference in a test, control reagents, apparatus for conducting atest, and the like. A second embodiment of a test kit comprises (a) anantibody as above, and (b) a specific binding partner for the antibodyconjugated to a detectable group. Ancillary agents as described abovemay likewise be included. The test kit may be packaged in any suitablemanner, typically with all elements in a single container along with asheet of printed instructions for carrying out the test.

In brief, in a particular embodiment of the present invention, blood iscollected and allowed to clot, and serum is removed. An aliquot of serumis placed into a reducing agent (e.g. dithiothreitol orβ-mercaptoethanol) to reduce the cysteines, thereby producing reactivesulfhydryl groups. The reduced serum is then incubated with an activatedsupport that reacts specifically and quantitatively with sulfhydrylgroups (an example of this support is the SULFOLINK® coupling gelproduced by Pierce Inc.). Incubation of reduced sera with this activatedsupport will therefore bind all of the ApoE molecules which containcysteine. After incubation, the serum containing unbound proteins isseparated from the activated support with its bound proteins (either bysedimentation or by centrifugation). The serum is then assayed for thepresence or absence of ApoE by an antibody specific for ApoE using anELISA format, with one of several possible detection schemes (e.g.colorometric). ApoE2 or ApoE3 will quantitatively bind to theactivated-support, and no free ApoE will be detected in the serum in theELISA. ApoE4 will not bind to the activated-support, and will thereforebe detected in the ELISA assay. Sera of a homozygote ApoE4 patient(containing only the ApoE4 isoform) will have about the same amount ofApoE in the sera after incubation with the activated-support as beforeincubation. Sera from a heterozygote ApoE4 patient (containing bothApoE4 and another ApoE isoform) will have approximately half the ApoEdetected after incubation with the activated-support as beforeincubation.

The present invention is explained in greater detail in the followingnon-limiting Examples in which ml means milliliters, μl meansmicroliters, and μg means micrograms.

EXAMPLE 1 Binding of Cerebrospinal Apolipoprotein E to ImmobilizedBeta-Amyloid Precursor Protein Fragments

βA peptides (Bachem), other peptides, or ethanolamine were covalentlyimmobilized to Immobilon AV affinity membrane (Millipore). The membraneis a chemically activated hydrophilic microporous membrane whichcovalently immobilizes peptides and proteins through amino and thiolgroups. βA peptides, or control peptides, were dissolved in distilledwater at one mg peptide/100 μL. Ten microliters (containing one hundredmicrograms peptide) were applied to a 13 mm diameter Immobilon disc, andincubated to dryness overnight at room temperature. Peptide was in largeexcess to the number of functional binding groups on the membrane.Control membranes were prepared by incubating the membrane in 2.0Methanolamine in 1.0M NaHCO₃, pH 9.5, to block the reactive groups on theImmobilon AV membrane. Membranes were stored at -20° C. in a desiccator.Prior to use the membranes were washed with phosphate buffered saline.The 12-28 Hydropathic Mimic Peptide and the Even-Hydro Peptide weresynthesized using standard f-Moc synthesis on an Applied Bio-Science430A peptide synthesizer. Amino acids were protected with blockinggroups, and were deprotected and washed prior to manual cleavage. Purityof the peptides was verified by reverse phase high performance liquidchromatography.

Binding of CSF proteins to immobilized peptides.

Immobilon AV membranes previously bound with βA peptides, withethanolamine, or with the hydromimic or even-hydro peptides wereincubated with 100 μL cerebrospinal fluid (previously filtered through a0.22μ filter) with 50 μL phosphate-buffered saline, pH 7.3, for thirtyminutes at room temperature. After incubation the membranes were placedin a Millipore filter holder (Swinnex) and washed with 3.0 mlphosphate-buffered saline, then washed with 700 μL 5% sodiumdodecylsulfate (SDS). The membranes were removed from the filter holder,cut in half and placed in 150 μL of Laemmli buffer (2% sodiumdodecylsulfate, 5% betamercaptoethanol, pH 6.8) and boiled five minutesto solubilize retained proteins. Forty-five μL of Laemmli buffer withsolubilized protein were loaded in each of ten lanes of a Bio-RadMinigel apparatus, with a stacking gel of 4%, and a separating gel of12% polyacrylamide with 2% SDS.

Immunodetection of ApoE.

Electrophoresed proteins were transferred to Immobilon P using standardWestern transfer techniques. After transfer the membrane was incubatedin Blotto (5% dried milk in Tris buffered saline, pH 7.6, with 0.5%Tween-20 (Pierce)) at room temperature for one hour. The membrane wasnext incubated in rabbit--anti-human apolipoprotein E antibody at1:1,000 dilution (kindly supplied by Dr. Joel Morrisett, Dept. ofMedicine, Baylor College of Med., Houston, Tex.) in Blotto overnight at4° C., then washed five times in Blotto. The membrane was exposed togoat --anti-rabbit secondary antibody conjugated with horse radishperoxidase at 1:10,000 dilution for one hour at room temperature, thenwashed seven times in Blotto. Horse-radish peroxidase was thenvisualized with Enhanced Chemiluminesce Detection kit (Amersham), andexposed to Hyperfilm ECL (Amersham).

Cerebrospinal fluid samples.

Cerebrospinal fluid was obtained from clinical diagnostic lumbarpunctures performed on human subjects following informed consent, andstored at -80° C.

Results.

Cerebrospinal fluid contains many proteins which bind to immobilized βApeptide, and to a control peptide ("even-hydro peptide") afterincubation and elution with phosphate buffered saline, FIG. 1 top,column 1. Among these retained proteins is apolipoprotein E, as shown inFIG. 1, bottom, column 1. Many of these proteins are eluted from bothimmobilized peptides by 5% sodium dodecyl sulfate, column 2; by 4 molarurea, column 3; or by 6 molar guanidine hydrochloride; column 4.Guanidine hydrochloride did not however elute apolipoprotein E from theβA.sub.(1-28) peptide, but eluted virtually all the apolipoprotein Epreviously bound to the control peptide, as shown in FIG. 1, bottom,column 4.

The binding of apolipoprotein E to various immobilized peptides is shownin FIG. 2. Apolipoprotein E in cerebrospinal fluid binds with highaffinity to immobilized βA.sub.(1-40), βA.sub.(1-28) and βA.sub.(12-28)following incubation and washing with PBS and 5% sodium dodecylsulfate.The "12-28 even hydro-peptide" contains the same amino acids asβA.sub.(12-28), but has a different hydropathic profile. ApolipoproteinE did not bind to this immobilized peptide, as shown in FIG. 2 bottom.The 12-28 hydropathic mimic peptide, contains different amino acids fromβA.sub.(12-28), but has a hydropathy profile very similar toβA.sub.(12-28), and also bound apolipoprotein E.

EXAMPLE 2 Immunodetection of ApoE in CSF of Alzheimer's and ControlPatients

Cerebrospinal fluid from both Alzheimer's disease patients, and controlpatients, contains immunoreactive apolipoprotein E. Apolipoprotein E ineach control CSF bound to the immobilized βA.sub.(1-28). ApolipoproteinE in the Alzheimer's disease cerebrospinal fluid samples, however,demonstrated marked variability of binding to the βA peptide.

EXAMPLE 3 Identification of ApoE Isoforms in Alzheimer's CSF byIsoelectric Focusing

To determine whether the variation in binding to the βA peptide notedabove could be explained by heterogeneity of the apolipoprotein Eitself, CSF proteins were resolved by isoelectric focusing andimmunoreactive apolipoprotein E was visualized. Cerebrospinal fluid(CSF) proteins were delipidated by agitation of 30 μL CSF with an equalvolume of chloroform:methanol :: 2:1 (V/V). Delipidated CSF was thenisoelectric focused using standard techniques on a urea, polacrylamidegel with Biorad ampholytes Ph 3-10 in a vertical slab gel apparatus(Biorad Minigel). Following electrophoresis, proteins were transferredto Immobilon P using Western technique and detected as described above.Several apolipoprotein E isoforms were detected by isoelectric focusing.

EXAMPLE 4 Detection of DNA Encoding the ApoE Type 4 Isoform in BloodCells of Alzheimer's Disease Patients

Population association studies of apolipoprotein E were carried out withover 85 blood samples from subjects afflicted with familial Alzheimer'sdisease, wherein the diagnosis of Alzheimer's disease wasautopsy-proven.

Genomic DNA from the blood samples was extracted in accordance withstandard techniques and amplified by polymerase chain reaction in eithera Stratagene SCS-96 or Techne MW-2 thermocycler using Techne HI-TEMP 96well plates and the primers described by P. Wenham et al., Lancet 337,1158 (1991). The polymerase chain reaction protocol was essentially asdescribed by Wenham et al. supra, and J. Hixson and D. Vernier, J. LipidRes. 31, 545 (1990). Each amplification reaction contained 20 ng genomicDNA, 1.0 pmol/μL each primer, 10% dimethylsulfoxide (Sigma), 200 μL eachdNTP (Pharmacia), 2.0 μCi (alpha-³² P) dCTP (800 Ci/mol in 10 mMTricine, NEN Research Products) 0.05 Units/μL Taq DNA polymerase andsupplied 1×incubation buffer (Boehringer Mannheim) in a final volume of15 μL. An initial denaturation at 94° C. for 5 minutes was followed by35 cycles of annealing at 65° C. for 0.5 minutes, extension at 70° C.for 1.5 minutes, denaturation at 94° C. for 0.5 minutes, and a finalextension at 70° C. for 10 minutes. After amplification, 5 units of HhaI(Pharmacia) were directly added to each well, and the plates wereincubated at least 3 hours at 37° C. Three μL of each reaction wereloaded on a 6% nondenaturing gel (0.4 mm thick ×43 cm long) andelectrophoresed for one hour under constant current (45 mA). Afterelectrophoresis, the gel was transferred to Whatman 3M chromatographypaper, dried, and autoradiographed for one hour using Kodak XAR-5 film.

Data are given in Table 1 below. The results showed that the otherwiseuncommon type 4 isoform was highly associated with Alzheimer's disease,compared to the normal population. The gene frequency of this type 4isoform of apolipoprotein E in the general population is 16%, while inthe Alzheimer's disease patients examined the gene frequency was 51%.

                  TABLE 1                                                         ______________________________________                                        ApoE Isotype in Normal and Alzheimer's Disease Patients.                      Apolipoprotein E                                                              Isotype (each  Patient Population                                             chromosome)    Alzheimer's Disease                                                                        Normal                                            ______________________________________                                        2/2              0%          1%                                               3/3            20.7%        60%                                               4/4            24.4%         3%                                               2/3             3.6%        12%                                               2/4             3.6%         2%                                               3/4            47.6%        23%                                               ______________________________________                                    

These data indicate that detection of DNA encoding the type 4 isoform ofapolipoprotein E is useful as a prognostic and diagnostic test forfamilial Alzheimer's disease.

EXAMPLE 5 Association of ApoE4 with Both Late-Onset Familial andsporadic Alzheimer's Disease

These data further support the involvement of the ApoE4 allele (ApoE-e4)in the pathogenesis of late-onset familial and sporadic AD.

Families.

Blood samples for the genomic DNA studies were obtained from familiesdescribed previously (J. Murrell et al., Science 254, 97-99 (1991); H.Karlinsky et al., Neurology 42, 1445-1453 (1992); P. St. George-Hyslopet al., Nature Genet. 2,330-334 (1992); M. Pericak-Vance et al., Am. J.Hum. Genet. 48, 1034-1050 (1991); and P. St. George-Hyslop et al.,Nature 347, 194-197 (1990)). All sampled individuals diagnosed asprobable Alzheimer's disease patients (AD) were examined by aneurologist and associated diagnostic personnel of the Joseph andKathleen Bryan Alzheimer's Disease Research Center (ADRC) MemoryDisorders Clinic at Duke University, the Centre for NeurodengenerativeDiseases at the University of Toronto, or the Departments of Neurologyat Massachusetts General Hospital and the Harvard Medical School. Theclinical diagnosis was made according to the NINCDS-ADRDA criteria (C.McKhann et al., Neurology 34, 939-944 (1984)). The Duke pedigrees wereprimarily late-onset AD families with an average age of 66.1±10.3 yearsin the 35 families. Three of the families could be classified asearly-onset (M<60 years) AD families. One family segregates with theAPP717valile mutation (A. Goate et al., Nature 349,704-706 (1991)), asecond segregates with the APP717val-phe mutation (J. Murrell et al.,Science 254, 97-99 (1991)), and the other is linked to chromosome 14markers with a maximum lod score of 3.5 (G. Schellenberg et al., Science258, 668-671 (1992) and P. St. George-Hyslop et al., Nature Genet 2,330-334 (1992)). The Toronto pedigrees were classified as primarilyearly-onset families (13 of the 17 families). Five of the families werelinked to chromosome 14 with lod scores greater than 3.0 in each family(P. H. St. George-Hyslop et al., Nature Genet 2, 330-334 (1992)) while asixth pedigree had the APP17valile mutation (H. Karlinsky et al.,Neurology 42, 1445-1453 (1992)). The family and genotypic data wereprocessed via the PEDIGENE™ system (C. Haynes et al., Genet. Epidemiol.3, 235-239 (1986)).

Genomic DNA from some patients diagnosed as sporadic cases of probableAD at Duke, Toronto, and Boston have been banked over the past sixyears. Sporadic AD patients were defined as those without a known familyhistory of AD or dementia. The sporadic, probable AD patientsrepresented all of the banked DNAs in the Toronto and Duke banks as ofNovember 1992, except for an ongoing prospective series begun in August1992 at the Bryan ADRC Memory Disorders clinic. The DNA from theseindividuals had been collected randomly prior to any interest in ApoEisotyping. Not all sporadic AD patients evaluated in these clinics werebanked routinely. The diagnosis of probable AD in this group can beexpected to be in the 80-90% accuracy range that is observed in mostspecialized AD clinics. Brain DNA was obtained from autopsy-confirmed,Caucasian cases of AD that had been banked in the Kathleen Bryan BrainBank at Duke, the University of Toronto, and the Harvard Medical School.Six black or American Indian autopsies were eliminated from the seriesof sporadic AD autopsies since the association analyses are sensitive tothe control group. Two sets of controls were used in the study. Thefirst set was 91 unrelated grandparents from the Centre d'Etude duPolymorphisme Humain (CEPH) reference families (J. Dausett et al.,Genomics 6, 575-577 (1990)). These families were collected for humangene mapping and are characterized by having grandparents, parents, andmany grandchildren available for DNA mapping. The grandparents representa random group of Caucasian aged-controls of European and Americanbackground, similar to the late-onset FAD families and theautopsy-confirmed sporadic AD population. Twenty-one Caucasian spousesof patients participating in an ongoing prospective analysis of probableAD patients and spouses were also used as control group.

Genomic DNA.

High molecular weight DNA was obtained from transformed lymphoblastsaccording to know techniques (M. Pericak-Vance et al., Neurology 36,1418-1423 (1986) and M. Pericak-Vance et al., Exp Neurol 102 271-279(1988)) or to the GENEPURE 341™ nucleic acid extractor's suppliedprotocol (Applied Biosystems). Genomic DNA from brain tissue wasisolated by pulverizing approximately 300 mg of frozen brain tissueunder liquid nitrogen, adding 4 ml of lysis buffer (Applied Biosystems)and 1 mg of proteinase K (Applied Biosystems), and gently rockingovernight at 37° C. before extracting on the GENEPURE 341™.

Amplification and restriction isotyping of ApoE.

Genomic DNA was amplified by polymerase chain reaction (PCR) in a TechneMW-2 thermocycler using HI-TEMP 96-well plates (Techne) and the primersdescribed by P. Wenham et al., Lancet 337, 1158-1159 (1991). The PCRprotocol was based on those described by. Wenham et al. and J. Hixson etal., J. Lipid Res. 31, 545-548 (1990). Each amplification reactioncontained 20 ng genomic DNA, 1.0 pmol/μl each primer, 10%dimethylsulfoxide (Sigma), 200 μm each DNTP (Pharmacia), 1.0 μCi(alpha-³² P) dCTP (800 Ci/mol in 10 mM Tricine, NEN Research Products),0.05 units/μl Taq DNA polymerase and supplied 1×buffer (BoehringerMannheim) in a final volume of 15 μl. An initial denaturation at 94° C.for 5 minutes was followed by 35 cycles of annealing at 65° C. for 0.5minutes, extension at 70° C. for 1.5 minutes, denaturation at 94° C. for0.5 minutes, and a final extension at 70° C. for 10 minutes. Afteramplification, 5 units of HhaI (Gibco) were directly added to each well,and the plates were incubated at least 3 hours at 37° C. Fifteen μL of2×Type III stop dye (J. Sambrook et al., Cold Spring Harbor LaboratoryPress B.24 (1989)) were added to each well, and 3 μL of each reactionwere loaded on a 6% nondenaturing polyacrylamide gel (0.4 mm thick×43 cmlong) and electrophoresed for one hour under constant current (45 mA).After electrophoresis, the gel was transferred to Whatman 3Mchromatography paper, dried, and autoradiographed for one hour usingKodak XAR-5 film. Each autoradiograph was read independently by twodifferent observers.

Statistical analvsis.

Allele frequencies for the control and AD groups were estimated bycounting alleles and calculating sample proportions. Allele frequencyestimates for the early- and late-onset FAD families were calculatedusing one randomly selected affected patient from each family. The Zstatistic for comparing two proportions was calculated (R. Elston and W.Johnson, Essentials of Biostatistics, (1987) and G. Schellenberg et al.,J. Neurogenet. 4, 97-108 (1987)). An extreme value of Z compared to theprobabilities for the standard normal distribution would suggestrejecting the null hypothesis that the allele frequencies in the twogroups are equal. In order to compare the ApoE allele frequencies in thedifferent populations, i.e., AD versus controls, and between controlgroups, the following comparisons were made: 1) CEPH controls versusspouse controls; 2) CEPH controls versus literature controls; 3) CEPHcontrols versus late-onset; 4) CEPH controls versus early-onset; 5) CEPHcontrols versus clinical (probable) sporadic AD; and 6) CEPH controlsversus autopsy-confirmed sporadic AD. Affected-pedigree-member (APM)linkage analysis for ApoE was analyzed in accordance with knowntechniques (M. Pericak-Vance et al., Am. J. Hum. Genet. 48, 1034-1050(1991) and D. Weeks et al., Am. J. Hum. Genet. 42, 315-326 (1988)). Twopoint lod scores were calculated using the computer program LINKAGE™Program Package (version 5.0) (G. Lanthrop et al., Am. J. Hum. Genet.36, 460-465 (1984) and G. Lanthrop et al., Proc. Natl. Acad. Sci. USA81, 3443-3446 (1984)). The age-of-onset and disease parameters used inthe lod score calculations were as formerly outlined (M. Pericak-Vanceet al., supra).

Results.

Table 2 illustrates the ApoE e4 allele frequency estimates in threecontrol populations: 1) 91 grandparents from the CEPH referencefamilies; 2) 21 spouses from an ongoing prospective study examiningconsecutive sporadic, probable AD patients, and 3) a representativecontrol series from a similar population in the literature (G. Lanthropet al., Proc. Natl. Acad. Sci. USA 81, 3443-3446 (1984)). Alsoillustrated are the ApoE e4 allele frequency estimates for severaldifferent Alzheimer's disease groups: 1) one randomly selected affectedindividual in the combined Duke and Toronto/Boston late-onset FADseries; 2) one randomly selected affected individual in the combinedToronto/Boston and Duke early-onset FAD families; 3) banked DNA samplesfrom sporadic patients carrying the diagnosis of probable AD from theDuke and Toronto/Boston clinics; and 4) DNA from 176 autopsy-confirmedsporadic AD patients from Duke and Toronto/Boston.

                  TABLE 2                                                         ______________________________________                                        ApoE-e4 Allele Frequency Estimates                                            Population    e4 Allele.sup.1                                                                           Z.sup.2 P Value                                     ______________________________________                                        Normal Controls                                                               CEPH.sup.3                                                                           (182)      0.16 ± 0.027                                             Spouses.sup.4                                                                         (42)      0.10 ± 0.046                                                                           0.59  0.56                                      Menzel.sup.5                                                                         (2000)     0.14 ± 0.008                                                                           0.71  0.48                                      Alzheimer's Disease (AD)                                                      LOAD.sup.6                                                                            (72)      0.42 ± 0.058                                                                           4.30  0.000017                                  EOAD.sup.7                                                                            (32)      0.19 ± 0.069                                                                           0.40  0.069                                     Clinical Sporadic                                                             AD.sup.8                                                                             (138)      0.36 ± 0.042                                                                           4.17  0.00031                                   Autopsy Sporadic                                                              AD.sup.9                                                                             (352)      0.40 ± 0.026                                                                           6.49  <0.00001                                  ______________________________________                                         .sup.1 Allele frequency estimates ± the standard error; number of          chromosomes counted is presented in parentheses.                              .sup.2 Z values are versus the CEPH control group.                            .sup.3 91 unrelated grandparents from the Centre D'Etude du Polymorphism      Humain (CEPH).                                                                .sup.4 Spouse controls in the Bryan ADRC Memory Disorders Clinic, Duke        University.                                                                   .sup.5 Population in H. Menzel et al., Apolipoprotein E polymorphism and      Coronary Artery Disease, Arteriosclerosis 3, 310-315 (1983).                  .sup.6 "LOAD" means Late Onset AD; One randomly selected affected from        each of 32 Duke and 4 Toronto/Boston lateonset FAD families.                  .sup.7 "EOAD" means Early Onset AD; One randomly selected affected from       each of 13 Toronto/Boston and 3 Duke earlyonset FAD families.                 .sup.8 39 Duke and 30 Toronto/Boston sporadic (probable) AD patients.         .sup.9 143 Duke and 33 Toronto/Boston autopsyconfirmed sporadic AD            subjects.                                                                

Consistent with Examples 1-4 above, the ApoE e4 allele frequency of therandomly selected affected patients in the predominantly late-onset FADfamilies was significantly different from that of the CEPH controls;0.50±0.06 versus 0.16±0.027 (allele frequency estimate±standard error,Z=2.44, P=0.014). Likewise, the combined Duke and Toronto/Bostonlate-onset FAD series presented here is significantly different from theCEPH controls (P=0.000017). The ApoE-e4 frequency of the combinedearly-onset FAD series from Toronto/Boston and Duke (0.19±0.069) did notdiffer significantly (P=0.069) from the frequency in the CEPH controls.Statistical analyses demonstrate highly significant differences in theApoE-e4 allele frequencies in both sporadic (probable) AD (P=0.00031)and autopsy-confirmed sporadic AD (P<0.00001) when compared to the CEPHcontrols.

EXAMPLE 6 Relation of Amyloid Beta-Peptide Deposition to ApolipoproteinE Type in Post-Mortem Tissue

In this Example, a series of brains from patients with sporadiclate-onset AD of known ApoE genotype was examined to determine whetherAD brains with an ApoE-e4 allele have a distinct pattern of amyloiddeposition. Brains of patients homozygous for ApoE-e4 were found tocontain increased vascular amyloid deposits and number and density ofamyloid and neuritic plaques compared to ApoE e3 homozygotes. Inimmunocytochemical studies, β-peptide was significantly increased inthose patients with one or two ApoE-e4 alleles. The three main ApoEgenotypes studied (e3/3, e3/4, and e4/4) showed no statisticallysignificant difference in sex, age of onset or duration of illness.Late-onset Ad cases associated with one or two ApoE-e4 alleles thus havea distinct neuropathological phenotype compared to cases homozygous forthe ApoE-e3 allele.

Case selection.

143 autopsy-confirmed cases of late-onset Alzheimer's Disease (AD)meeting NIH and CERAD criteria (C. McKhann et al., Neurology 34,939-944(1984) and S. Mirra et al., Neurology 41, 479-486 (1991)) and withoutaffected family members or other neurological disease were obtainedthrough the Kathleen Bryan Brain Bank at Duke University and analyzedfor ApoE genotype. These cases were banked between 1985 and 1992. Theaverage age at death for these 143 cases was 77.2±8.9 years of age withaverage duration of illness 8.6±4.2 years, and 63% of the patients werewomen.

ApoE Genotyping.

Genomic DNA was obtained by isolating DNA from approximately 300 mg offrozen brain tissue from each case. ApoE genotyping for each patient wascarried out as described above using amplification by polymerase chainreaction with ApoE primers and HhaI restriction isotyping withautoradiographic detection. ApoE genotyping demonstrated: 47 cases ApoEe3/3, 64 cases ApoE e3/4, 23 cases ApoE e4/4, 3 cases ApoE e2/3 and 6cases ApoE e2/4.

Neuropathological analysis.

Information on age at death, duration of illness, gender, brain weight,presence of amyloid deposits in cerebral vessels (using congo red stain)and description of neuritic plague density and amount of neurofibrillarytangles (modified King's silver stain B. Lloyd et al., J. Histotech 6,155-156 (1985)!) were taken from the original neuropathological reports.Neuritic plaque counts were mentioned in 100 cases and neurofibrillarytangle counts in 95 cases. Each count represented a single microscopicfield felt to be the most affected area in the section of that region.Plaques were counted with a 10×objective (field of 2.92 mm² whiletangles were counted with a 20×object (field of 0.72 mm²). Plaque countswere truncated at 100 per 10×field. All of this data had been enteredinto the patients medical record one to five years prior to ApoEgenotype analysis.

Analysis of congo red stained material for vascular amyloid.

A subset of 53 patients was chosen by selecting in chronological orderthose cases with preexisting congo red stained slides. Selection endedwhen 17 patients with ApoE genotype e4/4 (of the 23 total patients); 20patients with ApoE genotype e3/3 (of the 49 total patients); and 16patients with ApoE genotype e3/4 (of the 65 total patients) had beenchosen. Forty of the 53 autopsy reports specifically mentioned vascularamyloid and formed one set of blinded observations completed beforegenotyping. The presence of vascular amyloid was graded at three levels:no evident amyloid deposits (grade 0), trace amyloid deposits includingone positive vessel (grade 1), and readily identifiable vascular amyloid(grade 2). In addition, congo red stained slides of frontal cortex andhippocampus including entorhinal cortex from all 53 cases were examinedby three observers for vascular amyloid using the same grading system.These observations were analyzed independently and double-blind withaverage inter-rater agreement of 8.5%.

Immunocytochemical methods.

Paraffin blocks of hippocampal region, frontal lobe and parietal lobewere obtained for 7 cases of ApoE e4/4, 8 cases of ApoE e3/3, and 4cases of ApoE e3/4. Selection of cases was random without knowledge ofneuropathological report. 6-8 micron paraffin sections were cut andmounted on coated slides for immunocytochemistry. Sections weredeparaffinized, treated with 90% formic acid for 3 minutes, washed andthen incubated with monoclonal antibody for immunolocalization ofamyloid μ-peptide (antibody described in S. Ikeda et al., Prog. Clin.Biol. Res. 317,313-323 (1989), gift of Drs. George Glenner and DavidAllsop). Sections were reacted in parallel with identical antibodydilution and enzymatic detection steps using the ABC method(Vectorstain, Burlingame, Calif.), and a coverslip was placed overPermount after dehydration. Semi-adjacent sections (untreated withformic acid) were used to demonstrate neuritic plaques with monoclonalantibody (dilution 1:1000) to 164 kd-neurofilament protein (SMI-34,Sternberger-Meyer Immunocytochemicals, Inc) and for ApoE using apolyclonal antibody (dilution 1:20,000) to human ApoE which recognizesApoE2, ApoE3 and ApoE4 isoforms on Western blots (gift of Dr. JoelMorrisett, Baylor College of Medicine). Parallel controls were unstainedin these experiments.

Analysis of immunocytochemical material.

Regions of maximum plaque density in each section were selected andgraphical analysis was performed by drawing plaque outlines using acamera lucida onto a defined rectangle of 0.25 mm² divided into 160equal grids. Any gridboxes containing all or any portion of animmunoreactive plaque were counted, and the total was divided to actualgenotype. Inter-rate reliability is 85% with these methods, and variancebetween separate fields from the same section 15%. Representative fieldswere photographed with a Zeiss photomicroscope.

Statistical analysis.

Data were entered in a Statgraphics package and analysis of varianceused to describe the relationships between variables. No significantdifferences existed between the main set of 143 cases, the subset of 53cases, or the smaller subsets above of ApoE e3/3, e3/4, and e4/4genotypes chosen for congo red or immunocytochemical analysis withregard to age at death, gender ratio or brain weight. For comparison ofplaque and neurofibrillary tangle counts, Kruskal-Wallis one-wayanalysis was employed since observations did not fit a normaldistribution.

Immunolocalization of β-peptide amyloid deposits in brains of sporadicAD patients homozygous for ApoE e3 or e4.

Immunocytochemistry for β-peptide in brains of patients with sporadiclate-onset AD demonstrated consistent differences between immunostainedsections of cerebral cortex from ApoE e3/3 and ApoE e4/4 cases (data noshown). β-peptide immunoreactivity in the sections from ApoE e3/3 casesreveals minimal to absent vascular staining and faintly immunoreactiveplaques while sections from ApoE e4/4 cases are darkly stained withabundant immunoreactive vessels on the cerebral surface and stronglyimmunoreactive plaques and vessels in the parenchyma. The difference istypically of such magnitude that β-peptide immunostained sections fromApoE e4/4 brains can be differentiated from sections from ApoE e3/3brains without a microscope.

Immunolocalization of ApoE in brains AD patients homozygous for ApoE e3or e4.

ApoE immunoreactivity was observed in cerebral vessels, neurons, glialcells, senile plaques and neurofibrillary tangles as described inprevious reports (J. Diedrich et al., J. Virol. 65, 4759-4768 (1991);and Y. Namba et al., Brain Res. 541, 163-166 (1991)). Like β-peptideimmunoreactivity, ApoE immunoreactivity is enhanced after formic acidtreatment. There were no major differences in the localization orintensity of ApoE immunoreactivity in ApoE e3/3 cases compared to ApoEe4/4 (note that this polyclonal antiserum to ApoE detects both ApoE 3and ApoE 4 isoforms). In particular, most larger cerebral vessels wereApoE immunoreactive in both ApoE e4/4 cases and in ApoE e3/3 cases.

Extent of vascular amyloid in e3/3, e4/4 and e3/4 ApoEgenotypes--retrospective analysis of autopsy reports.

In 40 of the 53 cases, the presence or absence of vascular amyloiddetected by congo red staining was clearly noted in the autopsy report.Retrospective grading of these autopsy reports for amount of vascularamyloid deposits revealed a significant association of vascular amyloidwith number of ApoE e4 alleles (p<0.0001). Typically, no vascularamyloid was mentioned for ApoE e3/3 cases, trace vascular amyloid fore3/4 cases, and large amounts were observed in most e4/4 cases (Table3.A.1 below).

Prospective analysis of congo red stained material.

To examine prospectively the extent of vascular amyloidosis in the totalseries of 53 cases, a double-blind review of congo red stained sectionsof hippocampus and frontal cortex was conducted. This analysis confirmeda highly significant association between the presence of congo redpositive amyloid angiopathy and the dose of ApoE e4 allele in the 53cases (Table 3.A.1). Such amyloidotic vessels were not necessarilypresent throughout a given cortical region in ApoE e4/4 homozygotes, butoften predominated in the depths of sulci such as the hippocampalfissure.

Analysis of β-peptide immunoreactive vessels.

When sections from a subset of the series of 53 patients wereimmunoreacted for β-peptide and similarly rated, the same statisticallysignificant association between the amount of vascular amyloid and thedose of ApoE e4 allele was found (Table 3.C.1). Amyloidotic vessels inApoE e4 homozygotes included leptomeningeal vessels and large and smallvessels in the cortical plate with surrounding plaque-like accumulationof amyloid (plaque-like angiopathy of Scholze W. Scholze, Z. GesamteNeurol. Psych. 162, 694-715 (1938)!).

Neuritic plaque number is increased in ApoE e4 homozygotes compared toApoE e3 homozygotes.

In four out of five cortical regions, the average number of neuriticplaques in silver stained material was greater in ApoE e4 homozygotesthan in ApoE-e3 homozygotes (Table 1.A.2). Kruskal-Wallis one-wayanalysis revealed that increased average neuritic plaque count wassignificantly associated with ApoE-e4 allele dose in three regions:frontal, temporal and parietal cortex and that the association borderedon significance in the CA1 subfield of hippocampus. Neuritic plaquecounts did not correlate with duration of illness.

Neurofibrillary tangle counts are mildly increased in ApoE e4 comparedto ApoE e3 homozygotes.

In all five of the cortical regions presented above, the average numberof neurofibrillary tangles was greater in ApoE-e4 homozygotes comparedto ApoE-e3 homozygotes (Table 3.A.3). The chance that all five regionswould have increased average neurofibrillary tangle count in ApoE e4/4cases compared to ApoE e3/3 cases is 3% (nonparametric paired sampletest). The differences in average counts for each area are modest andnot significant by Kruskal-Wallis test. However, the averageneurofibrillary tangle count in each cortical region variessignificantly (p<0.01) with duration of illness in the whole set ofpatients and in the ApoE genotype subsets. The increase in averageneurofibrillary tangle counts in ApoE-e4 homozygotes is apparentlyaccounted for by the upward trend in duration of illness associated withApoE-e4.

Immunocytochemical analysis of amyloid deposition insporadic AD cases.

The large increase in amyloid deposition noted in ApoE-e4 homozygotescompared with ApoE-e3 homozygotes with sporadic AD includes bothvascular amyloid deposition and deposition in senile plaques. In orderto confirm qualitatively and quantitatively these differences, a set of7 patients homozygous for ApoE-e4 was compared to a set of 8 patientshomozygous for ApoE-e3. Four heterozygotes (ApoE-e3/4) were alamoexamined to determine possible ApoE dosage effects. Low power views ofcerebral cortex reveal the typical difference in over-all β-peptideimmunoreactivity observed in β-peptide immunoreacted cortical sectionsof ApoE e3 homozygotes compared to e4 homozygotes (date not shown).These differences are consistently found even when β-peptideimmunostained sections from a given cortical region are searched formicroscopic fields with maximum density of immunoreactive plaques.Plaques in ApoE e4/4 cases were more numerous, somewhat larger oftenincluding a vessel, and darker than those observed in ApoE e3/3 cases.

                  TABLE 3                                                         ______________________________________                                        Neuropathological and Immunochemical                                          Characteristics of Patients with Sporadic AD                                               ApoE GENOTYPE                                                                 e3/3   e3/4     e4/4                                             ______________________________________                                        Age at death: (yr)                                                                           76.4     79.1     75.8 NS                                      Illness duration: (yr)                                                                       7.7      8.5      9.0 p = .06                                  A. DATA FROM AUTOPSY REPORTS                                                  1. Congophilic amyloid angiopathy: Ave. grade (0 = none, 1 = trace,           2 = present)                                                                  By Report:     0.27     0.80     1.92 p < .0001                               By Review:     0.40     1.16     1.76 p < .0001                                              n = 20   n = 16   n = 17                                       2. Neuritic plaques: Ave. plaques per low power field w/10X                   objective (2.92 mm.sup.2)                                                     Frontal cortex 59       81       87 p < .0003                                 Temporal cortex                                                                              62       77       87 p < .009                                  Parietal cortex                                                                              61       68       86 p < .007                                  Entorhinal cortex                                                                            27       30       25 p = .69                                   Hippocampus (CA1)                                                                            12       13       18 p = .13                                                  n = 30   n = 48   n = 22                                       3. Neurofibrillary tangles: Ave. number per high power field                  w/20X objective (0.72 mm.sup.2)                                               Frontal cortex 6        7        8 p = .34                                    Temporal cortex                                                                              7        9        13 p = .16                                   Parietal cortex                                                                              7        10       8 p = .15                                    Entorhinal cortex                                                                            31       35       42 p = .22                                   Hippocampus (CA1)                                                                            26       37       48 p = .07                                                  n = 28   n = 46   n = 21                                       B. IMMUNOCYTOCHEMICAL STUDIES                                                 1. β-peptide immunoreactive vessels: Ave. grade (0 = none,               1 = trace, 2 = present)                                                                    0.50   0.50     2.00 p < .0001                                                n = 8  n = 4    n = 7                                            2. β-peptide immunoreactive plaques: % area occupied                     Strongly immunoreactive                                                                      4.5      9.4      31.7 p < .002                                Weakly immunoreactive                                                                        12.9     4.0      6.1 p = .04                                  Total plaques  17.4     13.4     37.8 p < .008                                               n = 8    n = 4    n = 7                                        ______________________________________                                    

Quantitative analysis of β-peptide immunoreactivity of plaques.

Sections of frontal cortex from fifteen cases of sporadic AD wereimmunoreacted for β-peptide localization and quantitative analysis wasmade of the microscopic fields containing maximum density of β-peptideimmunoreactive plaques for each case (Table 3.B.2). There was a highlysignificant, seven-fold greater average area covered by stronglyβ-peptide immunoreactive plaques in ApoE e4/4 homozygotes compared toApoE e3/3 homozygotes (p<0.002). ApoE e3/4 cases are intermediate.

In several ApoE e3/3 cases, weakly immunoreactive plaques were alsopresent and their number was significantly higher than in ApoE e4/4cases (p=0.04). Nevertheless, the average total area covered by allβ--peptide immunoreactive plaques was two-fold greater (p<0.008) in ApoEe4/4 homozygotes compared to ApoE e3/3 homozygotes (Table 3.B.2).

Extent of neuritic plaques.

Additional semi-adjacent section of frontal cortex from eight of theabove cases were immunostained with an antibody to neurofilament protein(SMI-34) to test whether neuritic plaque density varied between theApoE-e3 and ApoE-e4 homozygotes. SMI-34 immunoreactivity revealsneuritic processes and neurons with neurofibrillary tangles. Neuriticplaques can be identified by their content of neuritic processes.

Although the number and total area covered by neuritic plaques (asdefined by SMI-34 immunoreactivity) is less than that for amyloidplaques (as defined by β--peptide immunoreactivity) (data not shown),comparison of the plaque area measurements in semi-adjacent sectionsshows a significant and linear correlation for the two methods of plaquedetection in these eight cases (p<0.006, r=0.73). The area covered byneuritic plaques is also significantly greater in ApoE-e4 homozygotesthan in ApoE-e3 homozygotes (p<0.02).

EXAMPLE 7 Relationship of ApoE4 Gene Dose and Risk of Alzheimer'sDisease

This Example quantifies both the increased risk for AD and earlier ageat onset conferred by ApoE4 alleles in late onset AD families. Riskincreased from 20% to 90% and mean age at onset decreased from 84 to 68years with increasing number of ApoE4 alleles. These data indicate thatApoE4 gene dose is a major risk factor for late onset AD, and thathomozygosity for ApoE4 is virtually sufficient to cause AD by age 80.

Four of the 46 families tested to date are early onset families; twohave chromosome 21 APP mutations, and two are linked to chromosome 14.The frequency of ApoE4 was not elevated in these families. Members of 42late onset families diagnosed with AD or examined and found to beunaffected after age 60, with known ApoE genotype, were evaluated.Before enrollment in our study, informed consent was obtained from eachsubject or, when necessary, their legal guardian. All study protocolswere approved by the Duke University Medical Center Institutional ReviewBoard. Descriptive statistics for affected and unaffected subjects aregiven in Table 4. On average, women survived longer than men, whetheraffected (p=0.02) or unaffected (p=0.13).

With regard to Table 4, note that more than 90% of clinically diagnosedcases were confirmed at autopsy. The predictive value of clinicalexamination is nearly 100% when a family member has an autopsy-confirmeddiagnosis of AD. Four subjects with a diagnosis of AD and 30 subjectsexamined before age 60 were excluded from analysis. Late onset familieswere not linked to chromosome 21 or to chromosome 14 within 19%recombination of the region previously linked to early onset AD. Theestimated maximum two-point LOD score for AD and ApoE was 2.61 at 6%recombination.

                  TABLE 4                                                         ______________________________________                                        Descriptive Statistics for Affected and                                       Unaffected Subjects                                                           Affected Subjects                                                             (n = 95)                                                                             Age at Onset                                                                    n             Mean   (SD)                                            ______________________________________                                        Men      34            71.8   (8.4)                                           Women    61            70.4   (8.0)                                           ______________________________________                                        Unaffected Subjects                                                           (n = 139)                                                                            Age at Death                                                                    n             Mean   (SD)                                            ______________________________________                                        Men      62            77.7   (8.0)                                           Women    77            82.6   (9.2)                                           ______________________________________                                    

The proportion of affected individuals increased with the number ofApoE4 alleles from 20% of subjects with genotype 2/3 or 3/3, to 47% ofsubjects with genotype 2/4 or 3/4, and to 91% of subjects with the 4/4genotype (Table 5). This additive trend was highly significant(chi-squared=33.4 with 1 df, p <0.00001) (G. Koch et al., Analysis ofCategorical Data (Les Presses de L'Universite de Montreal, Montreal,1985) and SAS Institute, Inc., SAS/STAT Use's Guide, Release 6.03Edition (SAS institute, Cary N.C., 1988)). More women than men wereaffected (p=0.04). This suggests that women may possibly be at greaterrisk for AD.

                  TABLE 5                                                         ______________________________________                                        Percent Affected for Each ApoE Genotype                                       ApoE Genotype                                                                             % Men Affected (n)                                                                         % Women affected (n)                                 ______________________________________                                        2/2         -- (0)       -- (0)                                               2/3         28.6 (7)     11.1 (9)                                             3/3          7.1 (28)    28.6 (49)                                            2/4         50.0 (2)      0.0 (3)                                             3/4         38.3 (47)    54.5 (66)                                            4/4         91.7 (12)    90.9 (11)                                            ______________________________________                                    

As shown in Table 6, risk of AD increased by a factor of 2.84 (95%confidence interval (CI) 2.03 to 3.96) for each additional ApoE4 allele(D. R. Cox et al., Analysis of Survival Data (Chapman and Hall, London,1984) and L. Wei et al., JASA 84, 1065 (1989)). Hence, subjects with the4/4 genotype were more than 8 times as likely to be affected as subjectswith 2/3 or 3/3 genotypes. When separate estimates were made forsubjects with 0, 1, and 2 ApoE4 alleles, there was consistent evidence(which did not reach statistical significance) that women were at higherrisk than men. Risk for women over men was 1.33 (95% CI 0.42 to 4.26),1.39 (95% CI 0.76 to 2.55), and 1.30 (95% CI 0.50 to 3.38) at 0, 1, and2 ApoE4 gene doses, respectively.

                  TABLE 6                                                         ______________________________________                                        Percentage of Affected Subjects and Relative                                  Hazard According to the Number of ApoE4 Alleles.                              APoE4                                                                         GENE   Men        Women      Combined Hazard                                  DOSE   % Aff   (n)    % Aff (n)  % Aff (n)  Ratio                             ______________________________________                                        0      11.4    (35)   25.9  (58) 20.4   (93)                                                                              1.00                              1      38.8    (49)   52.2  (69) 46.6  (118)                                                                              2.84+                             2      91.7    (12)   90.9  (11) 91.3   (23)                                                                              8.07+                             ______________________________________                                    

Estimates of risk in Table 6 were derived by exponentiation of parameterestimates obtained from a Cox proportional hazard model which allowedrisk to differ in men and women (SAS Institute Inc., Analysis ofSurvival Data (Chapman and Hall, London, 1984), and SAS Institute Inc.,SAS Technical Report P-217, SAS/STAT Software: The PHREG Procedure,Version 6 (SAS Institute Inc., Cary N.C., 1991)). The symbol `+`indicates that hazard was significantly different from the referencevalue of 1. Information on each subject between the ages of 60 and 75was used as the assumption of proportional hazards did not hold afterthe diagnosis by age 75 of nearly all persons with 2 ApoE-e4 alleles.Statistically consistent, if not fully efficient, estimates of relativehazard result from proportional hazards models even when relatedindividuals are evaluated (L. Wei et al., JASA 84, 1065 (1989)). Thus,estimates of risk closely approximate estimates which would have beenfound by sampling just one person from each of a much larger collectionof families.

The Mantel-Haenszel correlation statistic (SAS Institute, Inc., SAS/STATUser's Guide, Release 6.03 Edition (SAS Institute, Cary N.C., 1988), andD. R. Cox et al., Analysis of Survival Data (Chapman and Hall, London,1984)), stratified by family, was used to evaluate the additive trend inrisk with increasing ApoE4 gene dose. The proportion of affectedsubjects increased significantly with ApoE4 gene dose (chi-sq=33.4 with1 d.f., p<0.00001).

Next examined was age at onset to determine if it was related to ApoE4gene dose. Onset distributions were constructed from information on ageat onset in affected subjects and age when last examined in unaffectedsubjects and were distinct for each gene dose (chi-sq=53.84 with 2 d.f.,p<0.00001) (R. G. Miller Jr., Survival Analysis (Whiley, N.Y., 1981),and SAS Institute Inc., SAS User's Guide: Statistics, Version 5 Edition(SAS Institute, Cary N.C., 1985)) (FIG. 3). Each additional ApoE4 alleleshifted onset to younger age; mean onset was 84.3 (SE 1.3) years insubjects with 1 ApoE4 allele, and 68.4 (1.2) years in subjects with 2ApoE4 alleles. Onset tended to be earlier in women than in men (p=0.04).

Similarly, survival distributions were constructed from information onage at death in subjects known to be deceased and from age when lastexamined in other subjects, regardless of affection status. ApoE4 genedose was related to survival (p=0.004); mean survival was 84.9 (SE 1.3)years in subjects with 0 ApoE4 alleles, 78.8 (SE 0.8) years in subjectswith 1 ApoE4 allele, and 78.1 (SE 1.4) years in subject with 2 ApoE4alleles. Earlier death in individuals with 1 or 2 ApoE4 alleles wasprimarily attributable to more frequent and earlier onset of AD in thesesubjects (p=0.001).

Despite shorter survival in subjects with 1 or 2 ApoE4 alleles, theearlier onset conferred by each ApoE4 allele leads us to suspect thatmost diagnoses of AD and most prevalent cases are in subjects with I or2 ApoE4 alleles. The difference between mean onset and mean survival was9.7 years in subjects with 2 ApoE4 alleles, 3.1 year in subjects with 1ApoE4 allele and 0.6 years in subjects with 0 ApoE4 alleles.

Previous reports of linkage of AD to markers near the ApoE locus mayhave resulted from the allelic association of AD with ApoE4. Althoughthese markers do not appear to be in disequilibrium with ApoE, theirsegregation could mimic linkage in families segregating at least oneApoE4 allele (D. A. Greenberg, Am. J. Hum. Genet. 52, 135-143 (1993)).The strong but not definitive evidence for linkage of AD to the ApoElocus (2=2.61, 0=0.06) also supports this explanation.

It is important to realize that 19 of 95 affected subjects in our cohortof pedigrees and 64 of 176 autopsy confirmed sporadic AD cases describedby Saunders et al. (R. G. Miller Jr., Survival Analysis (Wiley, N.Y.,1981)) had no ApoE4 alleles. Twelve of 42 late onset families hadaffected members with 0 ApoE4 alleles. The fact that these tended to bethe largest and based on simulation studies, the potentially mostinformative families for linkage, strongly suggests that other geneticsources of risk exist. These other genes will only be identified oncethe effects of the ApoE4 allele are included in subsequent analysis.

EXAMPLE 8 ApoE Isotyping Assay

400 μl of packed SULFOLINK® Resin (Pierce, Post Office Box 117,Rockford, Ill. 61105 USA) is washed with SULFOLINK® coupling buffer andresuspended in 2.0 ml of SULFOLINK® coupling buffer, all in accordancewith the manufacturer's instructions in the Pierce IMMUNOPURE® Ag/AbImmobilization Kit #2 (Pierce Catalog Number 44895G).

Four different assays were carried out, each in a 2 ml centrifugecolumn. Assay number 1 contained 180 μl of coupling buffer and 0.05 μgof purified human ApoE3. Assay number 2 contained 90 μl of resuspendedSULFOLINK® resin and 0.05 μg of purified human ApoE3. Assay number 3contained 180 μl of coupling buffer and 0.05 μg of purified human ApoE4.Assay number 4 contained 90 μl of resuspended SULFOLINK® resin and 0.05μg of purified human ApoE4.

Each assay was incubated at room temperature for 60 minutes. Each assaytube was then centrifuged 2 minutes in a desk top centrifuge and 30 μlof the supernatant was then withdrawn.

10 μl of Laemmli buffer (without β-mercaptoethanol) was added to eachsupernatant, and was placed in a well of a 12% polyacrylamide gel andelectrophoresed. The proteins were then transferred by Westerntechniques and the ApoE identified by an antiApoE antibody andvisualized using a secondary antibody and chemiluminescence detection,in accordance with known techniques (W. Strittmatter et al., Proc. Natl.Acad. Sci. USA 90, 1977-1981 (1993)).

Results are shown in FIG. 4, where "load" refers to the coupling bufferplus ApoE sample alone (assays 1 and 3) and "elute" refers to thesupernatant recovered from the resuspended SULFOLINK® resin assays aftercentrifugation (assays 2 and 4). Note that ApoE4 is found in the elute,while ApoE3 is not, demonstrating that the assay is capable ofdistinguishing between ApoE3 and ApoE4.

The foregoing is illustrative of the present invention, and are not tobe construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

What is claimed is:
 1. A kit for determining if a subject is atincreased risk of developing late onset Alzheimer's diseasecomprising:(A) at least one reagent that specifically detectsapolipoprotein E type 4 (ApoE4), wherein said reagent is selected fromthe group consisting of antibodies that selectively bind ApoE4, andoligonucleotide probes that selectively bind to DNA encoding ApoE4; and(B) instructions for determining that the subject is at increased riskof developing late onset Alzheimer's disease by(i) detecting thepresence or absence of an ApoE4 isoform in said subject with said atleast one reagent; and (ii) observing whether or not the subject is atincreased risk of developing late onset Alzheimer's disease by observingif the presence of ApoE4 is or is not detected with said at least onereagent, wherein the presence of ApoE4 indicates said subject is atincreased risk of developing late onset Alzheimer's disease.
 2. The kitof claim 1, wherein said at least one reagent and said instructions arepackaged in a single container.